CONSTRUCTION OF ESCHERICHIA-COLI VECTORS CONTAINING DEOXYADENOSINE AND DEOXYGUANOSINE ADDUCTS FROM (+)-ANTI-DIBENZ[A,J]ANTHRACENE DIOL EPOXIDE AT A DEFINED SITE

Dibenz[a,j]anthracene (DB[a,j]A) is a carcinogenic Polycyclic aromatic hydrocarbon, which is metabolically activated through the formation of bay region diol epoxides. Site-specifically modified M13mp19-based vectors containing a single (+)-anti-dibenz[a,j]anthracene diol epoxide {(+)-anti-DB[a,j]A-DE}-deoxyguanosine (dGuo) or -deoxyadenosine (dAdo) adduct were constructed. Four-base oligonucleotides, 5'-(HO)TGCA-3' and 5'-(HO)CATG-3', corresponding to the central four base pairs in the PstI and SphI restriction endonuclease sites, respectively, in the multiple cloning region of M13mp19, were reacted in solution with (+/-)-anti-DB[a,j]A-DE. The resulting adducted oligonucleotides were separated and purified using reverse-phase HPLC. Several different singly adducted oligonucleotides were isolated, consisting of the various cis and trans addition products of the (+) and (-) enantiomers of the diol epoxide bound to dGuo or dAdo in the oligonucleotides. 5'-(HO)TGCA-3' containing the (+)-anti-DB[a,j]A-trans-N2-dGuo adduct [T(DB[a,j]A-N2)GCA] and 5'-(HO)CATG-3' containing the (+)-anti-DB[a,j]A-trans-N6-dAdo adduct [C(DB[a,j]A-N6)ATG) were selected for subsequent ligation into M13mp19 vectors that had been constructed with a corresponding four base gap in the minus strand. Both unmodified and adducted oligonucleotides were successfully ligated into the M13mp19 vectors, [yields: unmodified -TGCA-M13mp19 (approximately 32%) and -CATG- M13mp19 (approximately 42%); adducted T(DB[a,j]A-N2)GCA-M13mp19 (approximately 13%) and C(DB[a,j]A-N6)ATG-M13mp19 (approximately 12%)]. The dAdo adduct-containing vector was characterized. The presence of a dAdo-DNA adduct at the recognition site of SphI inhibited restriction by SphI. The dAdo adduct-containing vector was cleaved with HindIII/XbaI to release the adduct in a 24-mer, which migrated slower than the corresponding 24-mer released from the unmodified vector. The dGuo adduct-containing vector was characterized similarly. While the results were more complicated, approximately 25% of the vectors contained the dGuo adduct. These site-specifically modified vectors will be used in future studies in vitro and in vivo to compare the effects of dAdo vs dGuo adducts on replication, repair, and mutagenesis.